1. Field of the Invention
This invention relates to the study of electrical activity of cells, and in particular the activity of nerve cells. Electrical potentials exist across the membranes of essentially all cells of the body, and some cells, such as nerve and muscle cells, are "excitable"--that is, capable of transmitting electrochemical impulses along their membranes.
The fluids located both inside and outside of nerve cells are electrolytic solutions. Generally, an excess number of negative ions accummulates immediately inside the cell membrane, and an equal number of positive ions accummulates immediately outside the membrane. The result of this is the development of what is known as a transmembrane potential. The transmembrane potential is believed to be primarily caused by the sodium "pump" in which positively charged sodium ions are transported to the exterior of the cell. Once the concentration of sodium outside the nerve cell has reached a particular value, sodium leaks back to the inside of the cell as rapidly as it is being pumped out, thus developing a state of equilibrium. Equilibrium occurs when the interior of the cell is approximately -85 millivolts with respect to the exterior of the cell.
So long as the membrane of the nerve cell remains completely undisturbed, the membrane potential remains at approximately -85 millivolts. This is called the resting potential. However, any factor that suddenly increases the permeability of the membrane to sodium is likely to elicit a sequence of rapid changes in membrane potential lasting a small fraction of a second, followed immediately by return of the membrane potential to its resting value. This sequence of changes in membrane potential is called the action potential.
Some of the factors that can elicit an action potential are electronic stimulation of the membrane, application of chemicals to the membrane to cause increased permeability to sodium, heat, cold or almost any other factor that momentarily disturbs the normal resting state of the membrane.
The action potential occurs in two separate stages called depolarization and repolarization. When the permeability of the cell membrane to sodium ions suddenly increases, many of the sodium ions rush to the inside of the cell, carrying enough positive charges to the inside to cause complete disappearance of the normal resting potential and usually enough charges to actually develop a positive state inside the cell instead of its normal negative state. This action is called depolarization and the positive state inside the cell is referred to as the reversal potential. Almost immediately after depolarization takes place, the pores of the cell membrane again become almost totally impermeable to sodium ions. Because of this, and because of potassium efflux resulting from an increase in potassium permeability, the reversal potential inside the cell disappears and the normal resting membrane potential returns. This is called repolarization. The change in membrane potential as the membrane permeability changes is shown in FIG. 1.
In order to study changes in membrane conductance, feedback circuits known as voltage clamps are utilized. Typically, the clamp will pass a variable current through the cell membrane such that the membrane voltage changes according to a preselected command voltage pattern in spite of changes of membrane conductance with voltage and time. The current is recorded and is used to compute membrane conductance at various voltage levels. By resolving changes in membrane conductance during excitation, especially conductance of sodium and potassium, the clamp allows resolution of subtle changes in membrane function including alteration in the sodium-potassium pump, and alteration of conductance-generating functions dependent upon membrane structure. In addition, changes in intracellular ionic concentrations relevant to cell function may be determined by clamping. The general level of cellular integrity and function can be determined best by clamping. Voltage clamp studies therefore are potentially very useful in the research of various neural diseases.
2. Description of the Prior Art
Early voltage clamp circuits employed a pair of microelectrodes which were inserted into the nerve cell under examination. A system of this type is shown in FIG. 2. The nerve cell comprises a cell body 10, dendrites 12, an axon 14 and nerve endings 16. Microelectrodes 18 and 20 are inserted through the cell membrane into the cell body. The electrode 18 is connected to voltage measurement circuitry 22 which measures the membrane potential and compares it to a command potential. An error feedback signal is generated as a function of the difference between the measured membrane potential and the command potential. The error feedback signal is used to control current injection circuitry 24, which injects current into the cell body via the electrode 20 so as to "clamp" the membrane potential to a value equal to the command potential. The amount of current necessary to achieve clamping is measured and can be used to determine conductance changes in the cell membrane.
The system shown in FIG. 2 thus utilizes separate electrodes for voltage measurement and current injection, and a continuous error feedback signal is provided. Although such a system provides very effective voltage clamping, the requirement of two electrodes makes it unusable in the study of many similar cells, particularly mammalian cells. In order to overcome this problem, voltage clamps utilizing a single electrode have been developed. In such circuits, the single microelectrode is rapidly switched from a current passing to a voltage recording mode. During a first portion of a cycle, the clamping circuitry is in a voltage recording mode and measures and stores the value of the membrane potential. The stored value is compared to the command potential. During the second half-cycle, the circuitry is switched to a current injecting mode and current is injected into the cell based upon the difference between the held value and the command potential. The single electrode clamp thus operates by providing discontinuous feedback, i.e., during the time that the membrane potential is being measured no clamping current can be injected. In order adequately to monitor the electrical activity of a cell, the clamp must be switched between the current injecting and voltage measuring modes at a rate which substantially exceeds the rates of conductance changes occuring within the cell. As can be seen in FIG. 1, conductance change which causes the membrane potential to change from the resting potential to the reversal potential can occur in a few tenths of a millisecond. In order to monitor such conductance changes, a switching frequency on the order of 100 kHz, or higher, is desirable.
Various single-electrode voltage clamps have been developed. Early clamps comprised a bridge circuit in which the cell membrane serves as an unknown resistor on one arm of a bridge. These clamps are inaccurate since the resistance of the electrode varies with current and time, thus making verification of bridge balance impossible, and thus resulting in errors in the measurement of membrane potential. A second type of single electrode clamp is disclosed in Wilson and Goldner, "Voltage Clamping With A Single Microelectrode", Journal of Neurobiology, Volume 6, No. 4, pages 411-422. In this circuit, the membrane potential at the output of an electrode preamplifier is sampled by sample-and-hold circuitry during periods when current injection is held to zero. The potential stored by the sample and hold is connected to a feedback circuit and compared with a command potential. During intervals of holding the membrane potential, the feedback circuit is switched to inject current based upon the difference between the held membrane potential and the command potential. The basic circuitry includes a high input impedance, low input capacity preamplifier to amplify the membrane voltage, sample-and-hold circuitry to store the amplified value, a current source to supply the necessary clamping current, a comparator for comparing the held membrane potential with the command potential and an FET switch to switch between the current injection and voltage measurement modes. Although this circuit is an improvement over the bridge-type circuit, its usefulness is limited in that its maximum switching rate is on the order of 10 kHz and minimum clamping time is greater than two milliseconds. This speed is insufficient to clamp fast events such as action potentials.
A specific preamplifier which may be used with a voltage clamp such as that disclosed in the Wilson et al. article is described in Kootsey and Johnson, "Buffer Amplifier With Femtofarad Input Capacity Using Operational Amplifiers", IEEE Transactions of Biomedical Engineering, Volume 20, September 1973, pages 389-391. This preamplifier provides an input capacity below 0.01 pF and an input resistance above 1012 ohms. A voltage clamp using a similar preamp is described in Merickel, "Design of a Single Electrode Voltage Clamp", Journal of Neuroscience Methods, 2 (1980) 87-101. This article discloses a circuit implementing the technique described in the Wilson et al. article. Since the clamping action takes about two milliseconds, this circuit is effective only for examining slow membrane activity, i.e., the circuit is too slow to be useful in the study of the action potential.